When imaging biological tissues, it is often desirable to enhance the signals measured from specific structures. Contrast agents, which produce a strong emission or reflection signal, have been utilized in virtually every imaging modality including ultrasound [2], computed tomography [57], magnetic resonance imaging [58], and optical microscopy [59].
Optical coherence tomography (OCT) is an emerging high-resolution medical and biological imaging technology [15–21]. OCT is analogous to ultrasound B-mode imaging except reflections of low-coherence light are detected rather than sound. OCT detects changes in the backscattered amplitude and phase of light.
Cross-sectional OCT imaging is performed by measuring the backscattered intensity of light from structures in tissue. This imaging technique is attractive for medical imaging because it permits the imaging of tissue microstructure in situ, yielding micron-scale imaging resolution without the need for excision and histological processing. Because OCT performs imaging using light, it has a one- to two-order-of-magnitude higher spatial resolution than ultrasound and does not require contact with tissue.
OCT was originally developed and demonstrated in ophthalmology for high-resolution tomographic imaging of the retina and anterior eye [22–24]. Because the eye is transparent and is easily optically accessible, it is well-suited for diagnostic OCT imaging. OCT is promising for the diagnosis of retinal disease because it can provide images of retinal pathology with 10 μm resolution, almost one order-of-magnitude higher than previously possible using ultrasound. Clinical studies have been performed to assess the application of OCT for a number of macular diseases [23,24]. OCT is especially promising for the diagnosis and monitoring of glaucoma and macular edema associated with diabetic retinopathy because it permits the quantitative measurement of changes in the retinal or retinal nerve fiber layer thickness. Because morphological changes often occur before the onset of physical symptoms, OCT can provide a powerful approach for the early detection of these diseases.
Recently, OCT has been applied for imaging a wide range of nontransparent tissues [16,17,25–27]. In tissues other than the eye, the imaging depth is limited by optical attenuation due to scattering and absorption. A “biological window” exists in tissue where absorption of near-infrared wavelengths is at a minimum and light can penetrate deep into highly-scattering tissue (FIG. 3) [28]. Because optical scattering decreases with increasing wavelength, OCT in nontransparent tissues has routinely used 1.3 μm wavelength light for imaging. In most tissues, imaging depths of 2–3 mm can be achieved using a system detection sensitivity of 110 dB (1 part in 1011). OCT has been applied to image arterial pathology in vitro and has been shown to differentiate plaque morphology with superior resolution to ultrasound [17,29].
Imaging studies have also been performed to investigate applications in gastroenterology, urology, and neurosurgery [30–32]. High resolution OCT using short coherence length, short-pulse light sources, has also been demonstrated and axial resolutions of less than 5 μm have been achieved [33,34]. High-speed OCT at image acquisition rates of 4 to 8 frames per second for 500 to 250 square pixel images has been achieved [35]. OCT has been extended to perform Doppler imaging of blood flow and birefringence imaging to investigate laser intervention [36–38]. Different imaging delivery systems including transverse imaging catheters and endoscopes, and forward imaging devices have been developed to enable internal body OCT imaging [39,40]. Most recently, OCT has been combined with catheter-endoscope-based delivery to perform in vivo imaging in animal models and human patients [41–44].
Apart from medical applications, OCT has been demonstrated as an emerging investigational tool for cell and developmental biology. OCT has imaged the development of numerous animal models including Rana pipiens and Xenopus laevis (Leopard and African frog), and Brachydanio rerio (zebrafish) [45–46]. High-speed OCT imaging has permitted the morphological and functional imaging of the developing Xenopus cardiovascular system, including changes in heart function following pharmacological interventions [47]. High-resolution imaging has permitted the real-time tracking of cell dynamics in living specimens including mesenchymal cell mitosis and neural crest cell migration [48]. OCT is advantageous in microscopy applications because repeated non-invasive imaging of the morphological and functional changes in genetically modified animals can be performed overtime without having to histologically process multiple specimens. The high-resolution, cellular-imaging capabilities suggest that OCT can be used to diagnose and monitor early neoplastic changes in humans.
The ability of OCT to perform optical biopsies, the in situ imaging of tissue microstructure at near-histological resolution, has been used to image morphological differences between normal and neoplastic tissue. OCT images of in vitro neoplasms of the female reproductive tract [49], the gastrointestinal tract [50], and the brain [51] have been investigated. Optical differences between normal and neoplastic tissue were evident, but primarily for late-stage changes. Still, situations exists where no inherent optical contrast exists between normal and pathologic tissue, such as in early-stage, pre-malignant tumors or in tumors which remain optically similar to normal tissue.
In the past, OCT has found numerous medical and biological applications. However, the imaging technique has relied largely on the inherent optical properties of the tissue to provide contrast and differentiate normal from pathological tissue. Phospholipid-coated perfluorobutane microbubbles (ImaRx Pharmaceutical, Tucson, Ariz.) have been used as a contrast agent for OCT; although they produce a strong OCT signal, blood and tissue also produce a fairly strong OCT signal, and the effects of this contrast agent in vivo on the visualization of blood vessels are subtle [60].
Albunex® is an FDA-approved, air-filled albumin composed of microparticles produced ultrasonically, that is used intravenously as an echo-contrast agent for echocardiography, and as a contrast agent for ultrasound imaging [2–4]. These microparticles may be formed with encapsulated liquid, to form a unique colloidal delivery vehicle. By the choice of protein used for the microparticle shell, the material encapsulated within the microparticle, a multitude of biomedical applications have been developed [3,5–9]. Some of the applications of these protein microparticles include biocompatible blood substitutes, magnetic resonance imaging and echocardiographic contrast agents, and novel drug delivery systems. These are described in the following U.S. Pat. Nos. 5,362,478; 5,439,686; 5,498,421; 5,505,932; 5,508,021; 5,512,268; 5,560,933; 5,635,207; 5,639,473; 5,650,156; 5,665,382 and 5,665,383.
These protein microparticles may be created from ultrasonic irradiation of aqueous protein solutions. Studies have delineated that the mechanism responsible for microparticle formation is, in fact, a combination of two acoustic phenomena: emulsification and cavitation. Ultrasonic emulsification creates the microscopic dispersion of the protein solution necessary to form the proteinaceous microparticles. Alone, however, emulsification is insufficient to produce long-lived microparticles. For example, emulsions produced by vortex mixing produce no long-lived microparticles.
Ultrasonic irradiation of liquids can also produce cavitation, the formation, growth, and implosive collapse of bubbles. The collapse of such bubbles creates transient hot-spots with enormous peak temperatures [14]. Sonolysis of water is known to produce H+, OH−, H2, H2O2, and in the presence of oxygen, HO2 [13]. Superoxide creates inter-protein disulfide bonds that cross-link the proteins and hold the microparticles together. This dispersion of gas or nonaqueous liquid into the protein solution, coupled with chemical cross-linking of the protein at the microparticle interface results in the formation of long-lived microparticles filled with air or nonaqueous liquid.